Oxymax chamber

Mouse metabolic rate was assessed by indirect calorimetry in open-circuit Oxymax chambers with CLAMS (Columbus Instruments) as previously described (Mitchell et al., 2018 (link)). In brief, mice were housed singly with water and food (WIS/NIA diet under AL, MF or CR feeding regimen) and maintained at ~24°C under a 12:12-h light-dark cycle (light period 0600–1800). All mice were acclimatized to monitoring cages for 3–6 h before recording began. Sample air was passed through an oxygen (O₂) sensor for determination of O₂ content. O₂ consumption was determined by measuring oxygen concentration in air entering the chamber compared with air leaving the chamber. The sensor was calibrated against a standard gas mix containing defined quantities of O₂, carbon dioxide (CO₂), and nitrogen (N₂). Constant airflow (0.6 L/min) was drawn through the chamber and monitored by a mass-sensitive flow meter. The concentrations of O₂ and CO₂ were monitored at the inlet and outlet of the sealed chambers to calculate oxygen consumption. Each chamber was measured for 30 s at 30-min intervals and data were recorded for 60 h total. Movement (both horizontal and vertical) was also monitored with beams that software transforms into counts of beam breaks by the mouse. Mice were 42 weeks of age, n = 5–6 per group.

After 5 weeks (Nprc^AKO) or 6 weeks (Nprc^MKO) on HFD, mice were housed and monitored seperately in open-circuit Oxymax chambers (Columbus Instruments) with free access to HFD and drinking water for 72 hours. Oxygen comsumption, carbon dioxide production, and physical activity were monitored at 15- to 20-min intervals. Food and water intake was also recorded. Data from the first 24 hours were discarded because the mice adapted to the new housing.